Peptide decoys for the preparation of medicaments intended for the prevention or treatment of autoimmune pathologies or disorders linked to the appearance of antibodies directed against exogenous proteins

ABSTRACT

Peptide or pseudopeptide decoys binding to antibodies capable of being developed against endogenous proteins within the framework of autoimmune pathologies or against exogenous proteins administered to patients, in particular within the framework of pathologies due to a deficiency in these proteins, the amino acid sequence of the peptide or pseudopeptide decoys differing from that of the epitopes recognized by the antibodies, for the preparation of medicaments intended for the prevention or treatment of the autoimmune pathologies, or for the prevention or treatment of the disorders linked to the appearance of antibodies directed against the exogenous proteins.

A subject of the invention is peptide decoys of pathogenic antibodiesfor the preparation of medicaments intended for the prevention ortreatment of autoimmune pathologies, or disorders linked to theappearance of antibodies directed against exogenous proteins which arerecombinant or non-recombinant used within the framework of thetreatment of these autoimmune pathologies or pathologies requiring theadministration of said exogenous proteins to patients.

Hemophilia A is a (severe) pathology due to the absence or insufficiencyof functional factor VIII (FVIII). The coagulation capacity is restoredin patients by the administration of concentrates of factor VIII whichis recombinant or originates from plasmas.

It is known that in a considerable percentage (approximately 15-25%) ofhemophilia A patients treated with factor VIII, an auto-immunizationprocess leads to the appearance of anti-FVIII antibodies inhibiting theprocoagulant function of the factor VIII administered, making thetreatment of the patients still more complex. These antibodies bind toseveral regions (or structural domains) of the FVIII protein, inparticular the C2, A2 and a3-A3-C1 domains.

The antibody response has the effect of inhibiting the procoagulantactivity of the FVIII administered and thus seriously complicating thetreatment of these hemophiliacs.

No current therapy completely resolves this problem; “immunologicaltolerization” by administration of massive doses of factor VIII has aconsiderable economic cost to the health system, which makes researchnecessary in order to develop more cost-effective processes.

It is known that antibodies have a specialized molecular structure,called a paratope, which allows them to perform an important biologicalfinction: the recognition of the antigen.

The paratope of the antibody is capable of recognizing not only alimited part (called the “epitope”) of the antigen which was at theorigin of its selection, but also smaller fragments of this antigen, inparticular peptides corresponding to the epitope (called “epitopicpeptides”), peptides possessing modifications of sequence or chemicalstructure relative to the epitope (called “epitope variants”) or alsomolecules not having sequence homology with the antigen (called“mimotopes”).

The antibody response against FVIII is polyclonal and heterogeneous inits specificity [Gilles et al., 1993, Blood, 2452-61] but it has beenobserved that the regions recognized by the inhibitor antibodies(epitopes) are restricted to a few principal zones of the factor VIIImolecule. The A2 domain and the C2 domain were initially identified byimmnunodot methods using proteolytic fragments of FVIII [Fulcher et al.,1985, PNAS USA 82, 7728-32] and subsequently confirmed when thenucleotide sequence of FVIII was determined (Vehar et al., 1984, Nature312, 337-342; Gitschier et al., 1984, Nature 312, 326-336) andrecombinant fragments prepared [Scandella et al., 1988, PNAS 86(4),1387; Scandella et al., 1989, Blood 74, 1618-26]. In one particularcase, the epitope of a murine monoclonal inhibitor antibody could berestricted to a sequence of 25 amino acids of the A2 region (residues484-508) thanks to the judicious use of hybrids between the sequences ofhuman and porcine factor VIII [Healey et al., 1995, J. Biol. Chem. 270,14505-9]. The cloning of a human antibody from B lymphocytes from ahemophilia patient made it possible to show that this antibodyrecognized the C2 domain and inhibited the FVIII function probably bybinding with a strong affinity to the binding site of the vonWillebrandt factor [Jacquemin et al., 1998, Blood 92, 496-506]. Finally,the a3-A3-C1 region also seems to be recognized by inhibitor antibodies.

The therapeutic approaches currently existing within the framework ofcombating the effects of these antibodies, are mainly the following:

-   -   stimulation by desmopressin of the production of FVIII in        moderate hemophiliacs [Kesteven et al., 1984, Thromb. Haemost.        52, 50-2];    -   activation of the coagulation cascade by prothrombin complex        concentrates [Lusher et al., 1983,Blood62, 1135-8];    -   administration of activated human recombinant FVII [Hedner et        al., 1993, Transfus. Med Rev. 7, 78-83];    -   use of porcine FVIII [Hay et al., 1996, Vox Sang 70, 68-9],    -   use of activated prothrombin concentrates (U.S. Pat. No.        4,160,02 (1979): Method for producing a        blood-coagulation-promoting preparation for human blood plasma;        U.S. Pat. No. 4,357,321 (1982): Method and composition for        treating clotting factor inhibitors; U.S. Pat. No. 4,663,164        (1987): Aqueous compositions for treating blood clotting        inhibitors),    -   use of factor VIII fragments (U.S. Pat. No. 4,649,132 (1987):        Treatment of factor VIII inhibitors; U.S. Pat. No. 4,769,336        (1987): Treatment of factor VIII inhibitors; U.S. Pat. No.        5,149,637 (1992): Recombinant factor VIII fragments),    -   use of immunocomplexes (U.S. Pat. No. 5,543,145 (1996):        Pharmaceutical composition and method for the suppression of        factor VIII inhibitor production),    -   use of hybrid human/porcine factor VIII molecules (U.S. Pat.        5,888,974: Hybrid human/animal factor VIII).

A conceptual approach of molecular decoys, not based on peptides orpeptide derivatives, has recently been described in two different modelsof human pathologies with humoral mediation: diabetes and myastheniagravis. In the two approaches, short sequences of RNA were selected(from a large library of RNA of random sequence) for their ability tobind specifically to a monoclonal antibody representative of the humanautoantibodies [Lee et al., 1997, Nat. Biotechnol. 15, 41-5; Lee et al.,1996, J. Exp. Med. 184, 315-24; Hay et al., 1996, mentioned above]. Inboth cases, the authors succeed in demonstrating that the decoysselected inhibit the binding of a few serums from patients to theirtarget (insulin receptor and acetylcholin receptor, respectively).However, these observations have not been validated in an animal modelof these pathologies. One of the weaknesses of this approach is themetabolic instability of the RNAs; although modified RNAs, moreresistant to the nucleases, have been used in certain of these studies,it may be thought that the plasma half-life of such molecules issufficiently weak to remove any chance for the decoy to act.

The present invention results from the demonstration by the Inventors,that peptides or pseudopeptides corresponding to epitope variants or tomimotopes, are capable of binding in the site of anti-FVIII antibodies,by preventing their binding to factor VIII, and thus reducing theanticoagulant activity of this antibody.

The present invention aims to provide new means for combating theantibodies present in the organism within the framework of autoimmunepathologies, or pathologies requiring the administration of proteins tothe patients, making it possible to considerably limit the cost of thecurrent therapies in this field, with an effectiveness comparable to oreven greater than current treatment methods.

A subject of the present invention is the use of peptide orpseudopeptide decoys binding to antibodies capable of being developedagainst endogenous proteins within the framework of autoimmunepathologies or against exogenous proteins administered to patients, inparticular within the framework of pathologies due to a deficiency inthese proteins, the amino acid sequence of said peptide or pseudopeptidedecoys differing from that of the epitopes recognized by saidantibodies, for the preparation of medicaments intended for theprevention or treatment of said autoimmune pathologies, or for theprevention or treatment of disorders linked to the appearance ofantibodies directed against said exogenous proteins.

A subject of the invention is more. particularly the abovementioned useof peptide or pseudopeptide decoys defined above, comprising betweenapproximately 6 and approximately 20 natural or non-natural amino acids(and preferably between approximately 8 and approximately 16 of theseamino acids), said decoys being selected for their ability to:

-   -   inhibit in vitro and in vivo the binding of antibodies to        proteins specifically recognized by the latter within the        framework of autoimmune pathologies, or pathologies requiring        the administration of said proteins to patients,    -   restore in vitro and in vivo the activity of said proteins in        the presence of said antibodies.

Advantageously, the antibodies preferentially recognize the peptide orpseudopeptide decoys rather than the abovementioned endogenous orexogenous proteins.

The invention also relates to the abovementioned use of decoys asdefined above, for the preparation of medicaments intended for theprevention or treatment:

-   -   of autoimmune pathologies such as:        -   myasthenia gravis,        -   the appearance of autoantibodies directed against insulin,        -   the appearance of autoantibodies directed against factor            VIII or IX within the framework of autoimmune variants of            hemophilia A or B respectively, in particular in pregnant            women in the case of hemophilia A,        -   the appearance of anti-sperm autoantibodies responsible for            certain cases of infertility,    -   or disorders linked to the appearance of antibodies directed        against exogenous proteins, or against the recombinant        endogenous proteins synthesized with a view to a gene therapy,        said proteins being administered within the framework of the        treatment of pathologies due to a deficiency in these proteins,        such as hemophilia A or B.

A subject of the invention is more particularly the use of decoys asdefined above, for the preparation of medicaments intended for theprevention or treatment of disorders linked to the appearance ofantibodies against endogenous factor VIII, or against exogenous factorVIII or derivatives, recombinant or non-recombinant, administered withinthe framework of hemophilia A.

The invention also relates to the abovementioned use of peptide decoysdefined above, as obtained from vectors expressing peptide libraries.

Therefore, a subject of the invention is more particularly theabovementioned use of decoys having no homology, or even a weak homologycomprised between approximately 10% and approximately 30%, with theepitopes of said proteins recognized by said antibodies, said decoysalso being designated mimotope decoys.

The invention relates more particularly to the abovementioned use ofmimotope decoys of factor VIII.

A subject of the invention is more particularly the abovementioned useof mimotope decoys of factor VIII of following formula (I):(X₁)_(n1)-C-Xa-C-(X₂)_(n2)  (I)in which:

-   -   n1 and n2 independently of one another represent 0 or 1    -   X₁ and X₂ independently of one another represent a natural or        non-natural amino acid,    -   X_(a) represents a chain of 8 to 10 natural or non-natural amino        acids.

The invention relates more particularly to the abovementioned use ofmimotope decoys defined above of formula (1) in which:

-   -   X₁ is chosen from Y, E, Q, S, R, A, K, N, or T,    -   X₂ is chosen from R, T, H, G, S, L, V, P, F, D, K, A, Q or I.

A subject of the invention is more particularly the abovementioned useof mimotope decoys defined above of formula (I) in which X_(a)represents the following sequence: NPSIGDKN SEQ ID NO: 1

Therefore, a subject of the invention is also more particularly theabovementioned use of the mimotope decoy of the following formula:YCNPSIGDKNCR SEQ ID NO: 2

The invention also relates to the abovementioned use of mimotope decoysof formula (I) in which X_(a) represents the sequence of the followingformula:(X₃)_(n3)-X₄-X₅-G-K-T-X₆-Lin which:

-   -   n3 represents 0 or 1,    -   X₃ represents any amino acid,    -   X₄ represents a hydrophobic amino acid,    -   X₅ represents an aromatic amino acid,    -   X₆ represents a hydrophobic amino acid.

A subject of the invention is more particularly the abovementioned useof mimotope decoys defined above of formula (I), characterized in that:

-   -   X₃ is chosen from I, Q, R, T, or K,    -   X₄ is chosen from V, T, L, I, M, F, W, or Y,    -   X₅ is chosen from F, or Y,    -   X₆ is chosen from A, M, Y, P, T; or V,

Therefore, the invention relates more particularly to the abovementioneduse of mimotope decoys defined above of the following formulae:ECIVYGKTALCT SEQ ID NO: 3 QCPTFGKTMLCT SEQ ID NO: 4 SCRLFGKTYLCH SEQ IDNO: 5 SCTVYGKTPLCG SEQ ID NO: 6 RCKTFGKTTLCS SEQ ID NO: 7 RCTVYGKTVLCLSEQ ID NO: 8

A subject of the invention is also the abovementioned use of mimotopedecoys of formula (I) in which X_(a) represents the sequence of thefollowing formula:X₇-X₈-W-X₉-N-R-X₁₀-X₁₁-(X₁₂)_(n12)-( X₁₃)_(n13)in which:

-   -   n12 and n13 independently of one another represent 0 or 1,    -   X₇ to X₁₃ represent any amino acid,    -   if appropriate W is replaced by F.

A subject of the invention is more particularly the abovementioned useof mimotope decoys defined above of formula (I), characterized in that:

-   -   X₇ is chosen from H, S, T, M, Q, or G,    -   X₈ is chosen from T, A, K, R, Q, or E,    -   X₉ is chosen from S, A, H, F, V, G, or T,    -   X₁₀ is chosen from R, K, L, S, H, T, I, or A,    -   X₁₁ is chosen from S, T, V, K, R, Y, M, or D,    -   X₁₂ is chosen from S, R, or L,    -   X₁₃ is chosen from I, H, or W.

Therefore, a subject of the invention is more particularly theabovementioned use of mimotope decoys defined above of formula (I)chosen from those of the following formulae: QCHTWSNRRSCL SEQ ID NO: 9SCHAWSNRRTCR SEQ ID NO: 10 RCHAWSNRKSCV SEQ ID NO: 11 CSKWANRLVSIC SEQID NO: 12 CSKWHNRSKRHC SEQ ID NO: 13 ACTTWSNRSKCP SEQ ID NO: 14ECTRWSNRSRCF SEQ ID NO: 15 CMKWSNRSSRWC SEQ ID NO: 16 KCGRWSNRSSCT SEQID NO: 17 CGRWFNRSDLHC SEQ ID NO: 18 ACHEWSNRSTCT SEQ ID NO: 19KCSRWTNRHLCD SEQ ID NO: 20 KCTRWTNRHLCS SEQ ID NO: 21 KCTRWTNRAHCP SEQID NO: 22 QCSKWVNRSRCA SEQ ID NO: 23 NCQKWTNRRTCL SEQ ID NO: 24QCGRWSNRSYCS SEQ ID NO: 25 TCHRWGNRTSCQ SEQ ID NO: 26 QCHRWANRISCS SEQID NO: 27 RCTQWTNRAYCP SEQ ID NO: 28 ACTQWSNRHMCG SEQ ID NO: 29TCHPFSNRSTCT SEQ ID NO: 30

A subject of the invention is also more particularly the abovementioneduse of the mimotope decoy of the following formula: SCHAWSNRRTCR SEQ IDNO: 10

A subject of the invention is also the abovementioned use of themimotope decoys of the following formula: KCEPDDPWPQCI SEQ ID NO: 31ACKRNHRWGACV SEQ ID NO: 32 ECGSHAWGRRCK SEQ ID NO: 33

The invention also relates to the abovementioned use of mimotope decoysdefined above of formula (I) in which X_(a) represents the sequence ofthe following formula:(X₁₄)_(n14)-X₁₅-X₁₆-H-X₁₇-W-G-X₁₈-(X₁₉)_(n19)in which:

-   -   n14 and n19 independently of one another represent 0 or 1,    -   X₁₄ to X₁₅ represent any amino acid.

The invention relates more particularly to the abovementioned use ofmimotope decoys defined above of formula (1), characterized in that:

-   -   X₁₄ represents K,    -   X₁₅ represents R, or G,    -   X₁₆ represents N, or S,    -   X₁₇ represents R, or A,    -   X₁₈ represents A, or R,    -   X₁₉ represents R.

Therefore, a subject of the invention is more particularly theabovementioned use of mimotope decoys defined above of formula (I)chosen from those of the following formulae: ACKRNHRWGACV SEQ ID NO: 34ECGSHAWGRRCK SEQ ID NO: 35

The invention also relates to the abovementioned use of peptide decoyscorresponding to epitopes recognized by the antibodies present in saidproteins, the peptide sequence of which is modified by suppression,substitution, or addition of at least one amino acid, said decoys beingdesignated epitope variants.

A subject of the invention is more particularly the abovementioned useof epitope variants of factor VIII.

A subject of the invention is still more particularly the abovementioneduse of epitope variants of factor VIII of the following formula (II):(C)_(n1)-X₁-X₂-X₃-L-T-D-S-E-M-D-V-V-R-X₄-X₅-(C)_(n2)  (II)in which:

-   -   n1 and n2 independently of one another represent 0 or 1,    -   X₁ to X₅ represent any amino acid.        with the exception of the peptide of formula DDDLTDSEMDVVRFD        (SEQ ID NO: 36).

The invention relates more particularly to the abovementioned use ofepitope variants defined above of formula (II), characterized in that:

-   -   X₁ represents D or A,    -   X₂ represents D or A,    -   X₃ represents D or A,    -   X₄ represents F, Y, or W,    -   X₅ represents D or A.

Therefore, a subject of the invention is more particularly theabovementioned use of epitope variants defined above of the followingformulae: DDDLTDSEMDVVRFD SEQ ID NO: 37 DDDLTDSEMDVVRYD SEQ ID NO: 38DDDLTDSEMDVVRWD SEQ ID NO: 39 CDDDLTDSEMDVVRFDC SEQ ID NO: 40AAALTDSEMDVVRFA SEQ ID NO: 41

The invention also relates to any pharmaceutical compositioncharacterized in that it comprises decoys as defined above, incombination with pharmaceutically acceptable vehicles.

Advantageously, the pharmaceutical compositions of the invention arepresented in forms capable of being administered by parenteral route orby oral route.

Preferably the dose of said pharmaceutical compositions is such that itis comprised between approximately 1 and approximately 5 mg/kg/day ofcompounds of formula (I) or (II) defined above.

The invention also relates to the peptide sequences of formula (I) asdefined.

A subject of the invention is also the peptide sequences of formula (II)as defined above.

The invention also relates to the products comprising:

-   -   at least one peptide sequence of formula (I) as defined above,        and/or at least one peptide sequence of formula (II) as defined        above,    -   and at least one compound chosen from the extracted, or        recombinant factor VIII, or other derived products of human or        animal origin,

as combination products to be used simultaneously, separately or spreadover time, in factor VIII substitution therapy, within the framework ofthe treatment or prophylaxia of hemorragic accidents in patientssuffering from hemophilia A.

A subject of the invention is also the pseudopeptides derived from thepeptide sequences of formula (I) and (II) defined above, in particularthe pseudopeptides obtained by conversion of one or more, or even all ofthe peptides of said peptide sequences to a D amino acid, which leads toa pseudopeptide considerably more resistant to proteolysis than theinitial peptide [Kramer et al., 1998, Protein Eng 11, 941-8].

As regards the synthesis of the abovementioned compounds of formula (I)or (II), the latter is advantageously carried out in the followingmanner.

In the procedure for solid phase synthesis of a polypeptide, the desiredpolypeptide is synthesized starting with an insoluble support such as abenzhydryl-amine or chloromethylated resin (derived from a cross-linkedpolystyrene, and available from suppliers of chemical products).

The amino acid at the C-terminal of the polypeptide to be synthesized,the a nitrogen of which as well as the reactive sites are protected byprotective groups, is bound to the resin using commonly-used strongcoupling techniques. The protective group of the amine function isdeprotected (leaving the other protective groups, if they exist, intact)in such a fashion that the following amino acid in the sequence (itselfalso having appropriate protective groups) is bound and so on. When thepolypeptide is entirely synthesized, it is separated from its resinsupport, and all the protective groups are eliminated, then thepolypeptide is recovered. Examples of appropriate protective groups:fluorenyl methyloxy carbonyl for the alpha amino, beta or gamma groups,tert-butyl ester for the aspartic and glutamic acids, tert-butyl oxycarbonyl for the lysine and tryptophan, trityl for the cysteine,asparagine, glutamine and histidine, tert-butyl for the serine andthreonine, 2,2,4,6,7-pentadihydrobenzo furan for the arginine.

The invention is further illustrated using the detailed descriptionwhich follows of the production of abovementioned compounds of formula(I) and (II), and of the demonstration of their ability to inhibit theanti-factor VIII antibodies.

I) Material and Methods

1) Techniques for Epitope-Variant-Type Peptide Decoys

Principle of the Spot Technique:

This parallel synthesis of peptides on Spot membrane (Frank, R. (1992)Spot Synthesis: an easy technique for the positionally addressable,parallel chemical synthesis on a membrane support, tetrahedron. 48,9217-9232) makes it possible to rapidly obtain a large number ofdifferent peptides brought together simultaneously on a plane cellulosesupport. The peptides are prepared on a scale of the order of 50 nmolper spot, in immobilized form. On a sheet of cellulose (12-8 cm)chemically reactive circular sites (spots) are created, approximately 5mm in diameter, which will serve as an anchorage point for binding theC-terminal amino acid of the peptide. The elongation of the peptidechain is carried out by successive additions of the amino acids of theC-terminal towards the N-terminal by esterification reaction. Theaminated functions of the residues used are temporarily protected duringcoupling by a labile group (a 9-fluorenylmethyloxycarbonyl or Fmocgroup) which is eliminated in basic medium. The reactive side chains areblocked by a stable group during the synthesis phase, and deprotected atthe end of assembly. A synthesis cycle takes place in 4 stages: firstlyformation of a peptide bond between the NH₂ of an amino acid alreadydeposited and the COOH of the following amino acid, which is activatedbeforehand by N-hydroxybenzotriazole and N,N diisopropylcarbodiirnide.Secondly, the free NH₂ functions not having reacted are acetylated inorder to avoid the formation of truncated peptides. Thirdly, the Fmocprotective group of the new amino acid is eliminated in basic medium bythe piperidine. Fourthly, the aminated functions thus obtained arelocated with bromophenol blue. This cycle is repeated as many times asnecessary. At the end of the synthesis, after having proceeded to thedeprotection of the side chains of the amino acids, the peptides whichremain bound in covalent manner to the membrane are analyzed by anindirect calorimetric immunoassay.

a) Preparation of the Membranes

The complete sequence of FVIII, with the exception of domain B, wassynthesized on cellulose membrane in the form of overlappingpentadecapeptides frameshifted by 2 residues according to the Spotmethod. The general protocol has been described previously by Molina etal. (Molina, F., Laune, D., Gougat, C., Pau, B. & Granier, C. (1996)Improved performances of spot multiple peptide synthesis, Pept Res. 9,151-5). The membranes are marketed by Abimed (Lagenfeld, Germany). TheFmoc amino acids and the N-hydroxybenzotriazole were ordered fromNovabiochem. The robot which carries out the different coupling stagesis an ASP222 robot (Abimed). All the peptides are acetylated on theirN-terminal. Once the peptides are assembled, the protective groups ofthe side chains are deprotected by a treatment with trifluororaceticacid.

b) Indirect Calorimetric Immunological Test

After saturation of the membrane in a blocking solution, the reactivityof the peptides is evaluated by immersion of the membrane in a solutionof inhibitor antibodies to be tested, generally used at a concentrationof 0.1-1 μg/ml. After washing, the peptide-antibodies interaction isdeveloped by incubation of an anti-Fc antibody marked with alkalinephosphatase used at 1:1000 (Sigma), which in the presence of itssubstrate (BCIP-MTT-MgCl₂ Sigma) produces a blue precipitate at thelevel of the peptides having bound the antibody to be tested. Themembranes are then regenerated in order to eliminate the blueprecipitate and the bound antibody with a view to other tests.

c) Production of Analogue Epitopes

Once the epitope is identified, a series of analogue peptides isprepared in Spot synthesis by substituting each position of the reactivesequence by the 19 amino acids (with the exception of cysteine). Theintroduction of a constraint by the addition of a sulphur bridge alsomakes it possible to obtain analogue epitopes.

2) Technique for “Mimotope”-Type Peptide Decoys

Principle of the Phase Display Technique

Peptide ligands can be easily obtained thanks to their ability to bindto an antibody by using the phage-display technique (Smith, G. P. (1985)Filamentous fusion phage: novel expression vectors that display clonedantigens on the virion surface, Science 228, 1315-7), based on thepresentation of random peptides at the surface of filamentous phages.These peptides can be exposed either at the surface of the pIII proteinof the phages (maximum number 5 copies) or at the surface of the pVIIIprotein (maximum number 2700 copies). These peptides, varying widely insize (from 6AA up to 30AA) can be either linear or constrained by asulphur bridge.

a) Different Peptide Phage Libraries Used

In order to carry out the screening of the random peptides, differentlibraries have been used:

-   -   4 peptide libraries, where the random peptide is expressed at        the surface of the pVIII of the phage (a linear 15^(mer), a        constrained 12^(mer), a semi-constrained 17^(mer) and a linear        30^(mer)) The latter, described by L.L.C. Bonnycastle        (University Burnaby, BC, Canada) (Bonnycastle, L. L.,        Mehroke, J. S., Rashed, M., Gong, X. & Scott, J. K. (1996)        Probing the basis of antibody reactivity with a panel of        constrained peptide libraries displayed by filamentous phage, J        Mol Biol. 258, 747-62), are constituted approximately by 10¹³        TU/ml and constructed starting with the f88.4 vector, derived        from the fd-tet phage.    -   2 peptide libraries, where the random peptide is expressed at        the surface of the pIII of the phage (a linear 9^(mer) and a        constrained 12^(mer)). The latter are described by Dr        Mazzuccheli L (Mazzucchelli, L., Burritt, J. B., Jesaitis, A.        J., Nusrat, A., Liang, T. W., Gewirtz, A. T., Schnell, F. J. &        Parkos, C. A. (1999) Cell-specific peptide binding by human        neutrophils, Blood 93, 1738-48), and their diversity is of the        order of 10⁹ TU/ml.

b) Screening of the Phage Library (Biopanning)

The biopanning technique is carried out following the abovementionedprotocol described by Smith et al. Three rounds of selection andamplification are carried out, in parallel for the pIII and pVIIIlibraries. For each selection cycle, the different inhibitor antibodies,at a concentration of 1-5 μg/ml for the first 2 rounds, 0.1-0.5 μg/mlfor the 3^(rd) round, in carbonate buffer (NaHCO₃, 100 mM, pH 8.6), areadsorbed on a 10×1.5 cm Petri dish (Falcon 1029) overnight at 4° C.under stirring. After five 2-minute washings with 0.05% TBS-T (TrisBuffered Saline: 1.37 M NaCl/26.8 mM KCl/0.5 M Tris base −0.05% Tween20/pH 7) in order to eliminate the excess of antibodies, thenon-specific sites are saturated for 2 hours at 37° C. with a solutionof 0.1% TBS-T −3% BSA previously filtered. After elimination of thislast solution by five 2-minute washings with 0.05% TBS-T, 2.10¹¹ TU(transduction units) of each primary library, i.e. 100 times thediversity of each library, or eluate of phages originating from thepreceding selection round are incubated overnight at 4° C. understirring. The phages which are not retained are eliminated by ten2-minute washings with 0.5% TBS-T followed. by five 2-minute washingswith 0.05% TBS-T. The phages retained are then eluted either by an acidelution (3 ml of HCl 0.1 M/glycine/BSA 1 mg/ml filtered previously/pH2.2, incubation of 30 min at AT under stirring, then neutralization with150 μl of 2 M Tris-HCl, pH 9) or by an elution by competition with FVIII(ON incubation at 4° C.). The different eluates are then used to infectE. Coli K91 cells for amplification. This amplification takes place in 2stages: firstly 5 ml of a bacterial suspension in exponential growthphase (absorbance 1.8 to 550 nm) is added to each eluate. Afterincubation for 10 minutes at 37° C. without stirring, 93 ml ofLuria-Bertani medium (LB) with 0.2 μg/ml of tetracycline (Tc) (pVIIIlibraries) or 0.75 μg/ml of kanamycin (Ka) (pIII libraries) are addedand incubated for 30 minutes at 37° C. at 225 rpm. The concentration isthen adjusted to 20 μg/ml for the Tc and 75 μg/ml for the Ka, then thesuspension is incubated at 37° C. at 225 rpm overnight. These differentbacterial suspensions are then centrifuged at 4° C. at 4000 rpm for 25minutes, then at 8000 rpm for 12 minutes, in order to precipitate thebacteria. The supernatant containing the phages is taken up inpoly(ethyleneglycol) 8000/2.5 M NaCl at a rate of 15 ml per 100 ml ofsupernatant; the precipitation is carried out overnight at 4° C. Aftercentrifugation at 4° C. at 8000 rpm for 40 minutes, the pellet is takenup in 3 ml of 50 mM TBS/NaCl, and incubated at 37° C. for 30 minutes at150 rpm. The content is transferred into 3 eppendorfs, centrifuged at15000 rpm for 10 minutes at 4° C. in order to eliminate the cell debris,then transferred into sterile 1.5-ml vials and stored at −80° C.

At each selection stage, the quantity of amplified phages is evaluatedby titration, whilst the enrichment with related phages is evaluated byELISA. After 3 selection rounds, 3 cloning stages made it possible toobtain on solid medium (LB-agar-Tc medium at 20 μg/ml or Ka at 75 μg/ml)isolated bacterial colonies. The ability of the different clones to bindspecifically to the inhibitor antibodies is then tested by ELISA.

c) Direct Elisa: Test of the Recognition of Inhibitor Antibodies by thePhages

The different inhibitor antibodies used for the screening of librariesare immobilized at 1 μg/ml in NaHCO₃ buffer on a microtitration plate(Nunc) overnight at 4° C. The wells are saturated with 0.1% PBS-T-2%milk for 1 hour 30 minutes at 37° C. The phages originating from thedifferent pannings, diluted to 1/25 in 0.1% PBS-T-2% milk buffer arethen incubated for 1 hour 30 minutes at 37° C. Between each of theseincubations, 3 washings with 0.1% PBS-T are carried out except aftersaturation. The bound phages are detected using an anti-M13 antibodycoupled to peroxidase (Amersham Boehringer) diluted to 1:3000, incubatedfor 1 hour at 37° C. After 5 washings, the peroxidase substrate (OPD) isadded. The reaction takes place over 30 minutes in the dark then anabsorbance measurement is carried out at 450 nm. After stopping thereaction by the addition of 4N H₂SO₄, a new measurement is carried outat 490 nm.

The specificity of the phages is demonstrated by the absence ofreactivity with irrelevant antibodies.

d) Determination of the Oligonucleotide Sequence

The sequencing of the DNA originating from the phages is carried out onan automatic sequencer (EUROGENTEC). The primers used: 5′ GTT TTG TCGTCT TTC CAG ACG 3′ for the pIII, and 5′ TCG GCA AGC TCT TTT AGG 3′ forthe pVIII, are localized downstream of the peptide insert.

3) Ability of the epitope or mimotope variant peptides in soluble formto inhibit their inhibitor antibodies in vitro and ex vivo

a) Synthesis of Soluble Peptides

The different epitopes identified in Spot or the mimotopes determined bythe phage display technique are then synthesized in the form of solublesynthetic peptides using an Abimed AMS422 synthesizer based on Fmocchemistry in soluble phase (Gausepohl, H., Boulin, C., Kraft, M. &Frank, R. W. (1992) Automated multiple peptide synthesis, Pept Res. 5,315-20). The peptides are deprotected and cleaved from the resin by atreatment with trifluoroacetic acid in the presence of appropriatescavengers. The peptides are then lyophilized, and their purity isevaluated by HPLC. If necessary, the peptides are purified in order toobtain more than 90% homogeneity by HPLC.

b) Neutralizing Ability of the Decoys In Vitro

The FVIII is immobilized on the ON plate at 4° C. In parallel, theinhibitor antibody is preincubated with its epitope peptide(s) in the ONrange at 4° C. in 0.1% PBS-T-2% milk. After saturation for 1 hour with0.1% PBS-T-2% milk, the Ab-peptide mixtures are incubated with theimmobilized FVIII for 2 hours at 37° C. Between each of theseincubations, 3 washings with 0.1% PBS-T are carried out except after thesaturation. Detection of the FVIII-Ab complex is achieved by a mouse orhuman anti-IgG coupled to peroxidase diluted to 1:3000 incubated for 1hour at 37° C. After 5 washings with 0.1% PBS-T, the peroxidasesubstrate (OPD) is added. The reaction takes place for 30 minutes in thedark then measurement of the absorbance is carried out at 450 nm. Afterstopping the reaction by the addition of H₂SO₄ 4N, a new measurement iscarried out at 490 nm.

c) Neutralizing Ability of the Decoys in a Functional Test (BethesdaTest)

This functional test (Kasper, C. K. & Pool, J. G. (1975) Letter:Measurement of mild factor VIII inhibitors in Bethesda units, ThrombDiath Haemorrh. 34, 875-6) consists of measuring the time necessary fora mixture composed of different coagulation factors (FIXa, FVIII,phospholipides, calcium etc.) in order to coagulate completely. Theinhibiting effect of an antibody is characterized in such a test by anextension of the coagulation time, the inhibiting ability of theantibody becomes greater as the extension of the coagulation timeincreases. The inhibiting character of an antibody is expressed inBethesda units, which corresponds to the reciprocal concentration ofantibodies in order to neutralize 50% of the coagulant activity of theFVIII. In contrast, the neutralizing effect of the peptides istranslated by a return to the initial coagulation time obtained in theabsence of inhibitor.

The functional test followed is derived from the abovementioned testdescribed by Kasper. The anti-FVIII monoclonal antibodies are diluted inan 0.05 M imidazole buffer, pH 7.3. An equal volume of this preparation(250 μl) with normal plasma buffered in imidazole (0.1 M, pH 7.4) aremixed and incubated for 2 hours at 37° C. Similarly, normal plasmabuffered in imidazole is mixed with a non-specific antibody whichconstitutes the control. The inhibiting activity of an antibody is readfrom a semi-logarithmic graph representing the correlation between theresidual activity of the FVIII and the inhibiting activity.

In order to demonstrate the ability of the peptides to neutralize theinhibitor antibodies, the inhibitor antibody used at a concentration soas to obtain 50% of residual FVIII, is preincubated ON at 4° C. inimidazole buffer with increasing peptide concentrations. This mixture isthen treated as above.

II) DETAILED DESCRIPTION

1) Choice of Antibodies:

The following were used:

-   -   a mouse monoclonal antibody, hereafter called ESH8, which has a        very considerable neutralization activity of the procoagulant        activity df the FVIII (6300 Besthesda Units/mg [Scandella et        al., 1995, Blood 86, 1811-9]). This antibody is a good        representative of the human antibodies binding to the C2 domain        of factor VIII,    -   a 2C11 human monoclonal antibody (Jacquemin et al., 1998,        abovementioned),    -   several mouse monoclonal antibodies, hereafter called F7B4,        F29F11, F14A12, F18B1, F19C2 and F21 C1 which represent a model        of the human antibodies binding to the a₁ acid region of factor        VIII for the first 3, and the a₃ acid region for the latter.

2) Processes for Obtaining Decoys:

“Epitope variant”-type peptide decoys were obtained, by using the Spottechnique in order to introduce variations of the epitope's amino acidssequence. The epitope variant peptides retain the reactivity with theantibody or can show a stronger reactivity than the epitope. Forexample, such an approach concerning the D³⁵⁶VVRF³⁶⁰ epitope of theanti-FVIII F7B4 antibody has shown that the F³⁶⁰ could be substitutedwithout loss of reactivity by the 2 other aromatic amino acids W and Y.In another example, the presentation of the epitope in cyclic form wasevaluated with the CDDDLTDSEMDVVRFDC peptide and the importance of thenegative charges was studied with the AAALTDSEMDVVRFA peptide. All these“epitope variant”-type decoys were obtained by standard peptidesynthesis and their binding activity to the anti-factor VIII antibodymeasured.

“Mimotope”-type peptide decoys were obtained by the recombinant phagelibrary technique. The approach of selection of peptides from randomlibraries of sequences has been described [Smith et al., 1993, MethodsEnzymol. 217, 228-257]. Peptides carried by the filamentous phages atthe surface of their envelope protein (PVIII or pIII) can be used: forexample, a library coding for peptides with 15 amino acids, and anothercoding for peptides with 12 amino acids, structurally constrained by theconstant presence of two cysteine residues. The conditions for selectionof the phages used are those described (in particular by [Ferrieres etal., 2000, Eur. J. Biochem. 267, 1819-1829]). The elution of the phagesspecifically captured by the antibody is carried either by acidtreatment or by displacement with an excess of antigen. The amino acidsequence of each peptide selected can be determined as described inparticular by Ferrieres et al. For example, mimotope peptides with theESH8 antibody, as well as with the 2C11 human antibody. could beobtained. Depending on the elution condition used, the consensus motifsobtained vary. By acid elution, two groups of sequences, all originatingfrom the constrained library of 12 amino acids, are obtained: the first,of mimotope type, comprises a single sequence YCNPSIGDKNCR, a sequencerepresented by 3 clones, the second group of epitope type isdistinguished by a consensus motif: X-C-X- hydrophobic-aromatic(Y/F)-G-K-T-X-L-C-X (where X represents any amino acid), which has ahomology of sequence with the C2 domain of factor VIII. This consensusmotif is found again in 6 different sequences: ECIVYGKTALCT (representedonce), QCQTFGKTMLCT (4 times), SCRLFGKTYLCH (once), SCTVYGKTPLCG (11times), RCKTFGKTTLCS (once) and RCTVYGKTVLCL (once). Whereas withelution with the antigen, a single sequence is obtained and itoriginates from the linear library of 15 amino acids KPGEVPRHRVTDFDR,represented 4 times.

In order to verify that these sequences taken outside the context of thephage are still reactive with the ESH8 antibody, these have beensynthesized on membrane by the Spot technique. It transpires that allhave retained their reactivity. Then, in order to determiner the keyresidues of the ESH8/peptide bond, each amino acid of the peptide wassubstituted by the other 19 amino acids (with the exception of cysteine)in order to determine 1) which were the residues critical to theESH8/peptide bond, 2) any tolerable substitutions which make it possibleto release a common characteristic (for example hydrophobic or aromaticcharacter, presence of a long side chain). The residues such as glycine(G), lysine (K), threonine (T) and leucine (L) do not tolerate anysubstitution, in contrast, with respect to the hydrophobic residue, themore reactive elements are valine (V), isoleucine (I) and leucine (L),but completely accept residues such as threonine (T), methionine (M),phenylalanine (F), tryptophan (W), and tyrosine (Y). With respect to thearomatic residue, this position mainly tolerates phenylalanine (F), andtyrosine (Y) with a slight acceptance of tryptophan (W).

Once these sequences are identified, a decoy is obtained by standardpeptide synthesis.

3) Processes for determining the blocking activity of the decoys:

The blocking activity of the decoys can be verified in vitro in an ELISAtest. The format used makes it possible to demonstrate the inhibition ofthe binding of the inhibitor antibody to factor VIII by the decoyintroduced in an increasing concentration. The neutralizing ability ofthe decoy can be confirmed in a functional test such as the BethesdaTest (method of Kasper, [Kasper et al., 1975, Thromb. Diath. Haemorrh.34, 875-6]).

The 7 soluble mimotope peptides more particularly studied are thefollowing: 103 KCGRWSNRSSCT 104 ECGSHAWGRRCK 105 CSKWHNRSKRHC 106CMKWSNRSSRWC 107 SCHAWSNRRTCR 108 QCHTWSNRRSCL 109 RCHAWSNRKSCV

The Inventors studied the ability of these soluble peptides to inhibitthe binding of the 2C11 antibody to factor VIII in an ELISA test invitro in the following manner: immobilization of FVIII, pre-incubationof 2C11 with the soluble peptides in the range (200 μM, 20 μM, 2 μM, 200nM, 20 nM, 2 nM) overnight at 4° C., then incubation of this premixtureon the FVIII, then development of the 2C11 antibody. The resultsobtained are indicated hereafter. % inhibition 103 104 105 106 107 108109 200 62.73 63.71 75.58 36.56 90.48 60.69 75.05 20 28.26 30.68 45.0421.99 80.04 25.99 37.48 2 3.47 13.44 10.27 0.00 37.33 15.79 12.46 0.02 00 0 0 0 0 0 0.002 0 0 0 0 0 0 0

All the peptides tested inhibit the 2C11 Ab/FVIII bond, peptide 107proving to be the most effective.

4) Ability of Peptide 107 (SCHAWSNRRTCR) to Neutralize the 2C11 Antibodyin Functional and In Vivo Analyses.

As peptide 107 has proved to be a peptide which is relatively effectiveat inhibiting the FVIII-2C11 antibody bond, the Inventors have taken amore detailed interest in its ability to neutralize the inhibitingactivity of 2C11.

Firstly, the Inventors evaluated in a functional coagulation test(Bethesda test) the ability of peptide 107 to restore the procoagulantactivity of FVIII in the presence of 2C11. FIG. 2 depicts thedose-dependent neutralization of the inhibiting activity of 2C11 bypeptide 107. At a 2C11 concentration of 3.5 nM, a concentration at which98% of the activity of the FVIII is inhibited (FIG. 1), the totalneutralization of its inhibiting effect is. achieved with a peptide 107concentration of the order of 100 μM, the IC₅₀ being equal to 19 μM. Acontrol peptide used in an identical concentration range has noneutralizing effect (FIG. 2). Peptide 107 incubated with FVIII alonedoes not at all modify the procoagulant activity of the FVIII (FIG. 2).The peptides (103, 104, 105, 106, 108, and 109) can also effectivelyneutralize the inhibiting activity of 2C11. However, higher peptideconcentrations are necessary in order to achieve an effectivenesssimilar to that of peptide 107 (data not shown).

Secondly, the neutralization properties of peptide 107 were studied invivo, using a mouse model deficient in FVIII. A preliminary study madeit possible to specify that an intravenous administration of 0.5 UI ofhuman recombinant FVIII to such mice made it possible to obtain an FVIIIplasma count of the order of 0.3 IU/ml FVIII, a count which is stablefor at least one hour. Having determined this, 2C11 antibody (16.7 nM)incubated alone, in the presence of peptide 107, or a control peptide(650 μM and 700 μM, respectively) was injected into the tail vein ofthree groups of mice respectively. Thirty minutes later, the FVIIIplasma count of these mice was reconstituted by the injection of 0.5 UIof human FVIII and after fifteen minutes the plasma procoagulantactivity was measured. FIG. 3 proves that the administration of 2C11alone completely inhibits the procoagulant activity of the FVIIIinjected, whereas the mice which have received the 2C11 antibody in thepresence of peptide 107 maintain 52% of the procoagulant activity of theFVIII. The neutralizing activity of peptide 107 is very specific, sincea control peptide has no neutralizing effect.

Therefore, these data indicate that the ability of peptide 107 toneutralize the inhibition of the 2C11 antibody vis-à-vis FVIII,suggested by in vitro experiments, is also verified under in vivoconditions, which resemble hemophilia A.

Thirdly, the Inventors looked at whether the neutralizing properties ofpeptide 107 vis-à-vis 2C11 could also be applied to other serums ofhemophilia patients with inhibitors. A preliminary study reveals thatout of 12 serums of hemophilia patients with inhibitors, 2 of them reactwith peptide 107 thus indicating that FVIII inhibitors similar to 2C11are produced by other patients with hemophilia A.

FIG. 1

FIG. 1 represents the effect of the 2C11 antibody on the activity offactor VIII evaluated by the Bethesda test. Factor VIII was incubated inthe presence of 3.5 nM of 2C11 antibody for 2 hours at 37° C., theresidual activity of factor VIII was then measured with a coagulometer,in one stage.

The percentage of residual activity of factor VIII is represented on theY-axis. Bar A represents the activity of the factor VIII alone (100%),Bar B represents the factor VIII activity in the presence of the 2C11antibody at a concentration of 3.5 nM.

In the presence of 3.5 nM of 2C11 factor VIII activity is inhibitedalmost completely.

FIG. 2

FIG. 2 represents the evolution of the activity of factor VIII,evaluated by the Bethesda test, in the presence of 3.5 nM of 2C11antibody as a function of peptide concentration. The 2C11 antibody wasincubated overnight at 4° C. in the presence of a control peptide orpeptide 107 (SCHAWSNRRTCR) in variable concentration. The factor VIIIwas then incubated for 2 hours at 37° C. with these peptide-antibodymixtures or with peptide 107 alone, then its residual activity wasmeasured with a coagulometer, in one stage.

The percentage of residual activity of factor VIII is represented on theY-axis, the peptide concentration is represented on the X-axis (μM). Theblack triangles represent the evolution of the residual activity offactor VIII in the presence of increasing concentrations of peptide 107alone, the black dots represent the evolution of the residual activityof factor VIII in the presence of the mixture of increasingconcentrations of peptide 107 and the 2C11 antibody, the white diamondsrepresent the evolution of the residual activity of factor VIII in thepresence of the mixture of increasing concentrations of the controlpeptide and the 2C11 antibody.

Peptide 107 makes it possible to neutralize the effect of the 2C11antibody.

FIG. 3

FIG. 3 represents the effect of the injection of the 2C11 antibody inthe absence or in the presence of peptide 107 or of a control peptide onthe blood coagulation of mice deficient in factor VIII and to whichhuman factor VIII is administered.

The 2C11 antibody (16.5 nM) or a mixture containing the 2C11 antibody(16.5 nM) and peptide 107 (650 μM) or a control peptide (700 μM) wasinjected into the tail vein of a “knock out” mouse, deficient in factorVIII, at a rate of 2.5 μg of 2C11 antibody, 1 mg of peptide 107 and 1 mgof control peptide. After 30 minutes, 0.5 UI of human factor VIII wasinjected and the plasma procoagulant activity was then measured by achromogenic test 15 minutes later.

The percentage of residual activity of factor VIII is represented on theY-axis. Bar A represents the effect of the injection of factor VIIIwithout prior injection of antibodies (100% of residual activity). Bar Brepresents the effect of the injection of the 2C11 antibody alone priorto the injection of factor VIII. Bar C represents the effect of theinjection of a mixture of 2C11 antibody and peptide 107 prior to theinjection of factor VIII. Bar D represents the effect of the injectionof a mixture of the 2C11 antibody and a control peptide prior to theinjection of factor VIII.

Peptide 107 specifically neutralizes the effect of the 2C11 antibody invivo.

1. A method for the prevention or treatment of actoimmune pathologies,or for the prevention or treatment of disorders linked to the appearanceof antibodies directed against exogenous proteins in a subject in needthereof comprising administering to said subject an effective amount ofpeptide or pseudopeptide decoys binding to antibodies capable of beingdeveloped against endogenous proteins within the framework of autoimmunepathologies or against exogenous proteins administered to the patients,in particular within the framework of pathologies due to a deficiency inthese proteins, the amino acid sequence of said peptide or pseudopeptidedecoys differing from that of the epitopes recognized by saidantibodies.
 2. The method according to claim 1, wherein said peptides orpseudopeptide decoys comprise between approximately 6 and approximately20 natural or non-natural amino acids (and preferably betweenapproximately 8 and approximately 16 of these amino acids), said decoysbeing selected for their ability to: inhibit in vitro and in vivo thebinding of antibodies to proteins specifically recognized by the latterwithin the framework of autoimmune pathologies, or pathologies requiringthe administration of said proteins to patients, restore in vitro and invivo the activity of said proteins in the presence of said antibodies.3. The method according to claim 1, wherein said subject is sufferingfrom or exhibits myasthenia gravis, the appearance of autoantibodiesdirected against insulin, the appearance of autoantibodies directedagainst factor VIII or IX within the framework of autoimmune variants ofhemophilia A or B respectively, in particular in pregnant women in thecase of hemophilia A, the appearance of anti-sperm autoantibodiesresponsible for certain cases of infertility, or disorders linked to theappearance of antibodies directed against exogenous proteins, or againstthe recombinant endogenous proteins synthesized with a view to a genetherapy, said proteins being administered within the framework of thetreatment of pathologies due to a deficiency in these proteins, such ashemophilia A or B.
 4. The method according to claim 1, wherein saidsubject suffers from a disorder linked to the appearance of antibodiesagainst endogenous factor VIII, or against exogenous factor VIII orderivatives, recombinant or non-recombinant, administered within theframework of hemophilia A.
 5. The method according to claim 1, whereinsaid peptide decoys are obtained from vectors expressing peptidelibraries.
 6. The method according to claim 1, further comprisingadministering decoys having no homology, or even a weak homologycomprised between approximately 10% and approximately 30%, with theepitopes of said proteins recognized by said antibodies, said decoysalso being designated mimotope decoys.
 7. The method according to claim1, further comprising administering mimotope decoys of factor VIII. 8.The method according to claim 7, wherein said mimotope decoys of factorVIII have the following formula (I):(X₁)_(n1)-C-X_(a)-C-(X₂)_(n2) in which: n1 and n2 independently of oneanother represent 0 or 1, X₁ and X₂ independently of one anotherrepresent a natural or non-natural amino acid, X_(a) represents a chainof 8 to 10 natural or non-natural amino acids.
 9. The method accordingto claim 8, wherein said mimotope decoys of formula (I) in which: X₁ ischosen from Y, E, Q, S, R, A, K, N, or T, X₂ is chosen from R, T, H, G,S, L, V, P, F, D, K, A, Q or I.
 10. The method according to claim 9,wherein said mimotope decoys of formula (I) wherein X_(a) represents thefollowing sequence: NPSIGDKN SEQ ID NO: 1


11. The method according to claim 10, wherein said mimotope decoy hasthe following formula: YCNPSIGDKNCR SEQ ID NO: 2


12. The method according to claim 8, wherein said mimotope decoys offormula (I) wherein X_(a) represents the sequence of the followingformula: (SEQ ID NO: 48) (X₃)_(n3)-X₄-X₅-G-K-T-X₆-L

in which: n3 represents 0 or 1, X₃ represents any amino acid, X₄represents a hydrophobic amino acid, X₅ represents an aromatic aminoacid, X₆ represents a hydrophobic amino acid.
 13. The method accordingto claim 12, wherein said mimotope decoys are characterized as: X₃ ischosen from I, Q, R, T, or K, X₄ is chosen from V, T, L, I, M, F, W, orY, X₅ is chosen from F, or Y, X₆ is chosen from A, M, Y, P, T, or V, 14.The method according to claim 12, wherein said mimotope decoys are ofthe following formulae: ECIVYGKTALCT SEQ ID NO: 3 QCPTFGKTMLCT SEQ IDNO: 4 SCRLFGKTYLCH SEQ ID NO: 5 SCTVYGKTPLCG SEQ ID NO: 6 RCKTFGKTTLCSSEQ ID NO: 7 RCTVYGKTVLCL SEQ ID NO: 8


15. The method according to claim 8, wherein said mimotope decoys offormula (I) wherein X_(a) represents the sequence of the followingformula:X₇-X₈-W-X₉-N-R-X₁₀-X₁₁-(X₁₂)_(n12)-(X₁₃)_(n13) in which: n12 and n13independently of one another represent 0 or 1, X₇ to X₁₃ represent anyamino acid, if appropriate W is replaced by F.
 16. The method accordingto claim 15, wherein mimotope decoys are characterized as: X₇ is chosenfrom H, S, T, M, Q, or G, X₈ is chosen from T, A, K, R, Q, or E, X₉ ischosen from S, A, H, F, V, G, or T, X₁₀ is chosen from R, K, L, S, H, T,I, or A, X₁₁ is chosen from S, T, V, K, R, Y, M, or D, X₁₂ is chosenfrom S, R, or L, X₁₃ is chosen from I, H, or W.
 17. The method accordingto claim 15, wherein said mimotope decoys are chosen from those of thefollowing formulae: QCHTWSNRRSCL SEQ ID NO: 9 SCHAWSNRRTCR SEQ ID NO: 10RCHAWSNRKSCV SEQ ID NO: 11 CSKWANRLVSIC SEQ ID NO: 12 CSKWHNRSKRHC SEQID NO: 13 ACTTWSNRSKCP SEQ ID NO: 14 ECTRWSNRSRCF SEQ ID NO: 15CMKWSNRSSRWC SEQ ID NO: 16 KCGRWSNRSSCT SEQ ID NO: 17 CGRWFNRSDLHC SEQID NO: 18 ACHEWSNRSTCT SEQ ID NO: 19 KCSRWTNRHLCD SEQ ID NO: 20KCTRWTNRHLCS SEQ ID NO: 21 KCTRWTNRAHCP SEQ ID NO: 22 QCSKWVNRSRCA SEQID NO: 23 NCQKWTNRRTCL SEQ ID NO: 24 QCGRWSNRSYCS SEQ ID NO: 25TCHRWGNRTSCQ SEQ ID NO: 26 QCHRWANRISCS SEQ ID NO: 27 RCTQWTNRAYCP SEQID NO: 28 ACTQWSNRHMCG SEQ ID NO: 29 TCHPFSNRSTCT SEQ ID NO: 30


18. The method according to claim 17, wherein the mimotope decoy is ofthe following formula: SCHAWSNRRTCR SEQ ID NO: 10


19. The method according to claim 8, wherein the mimotope decoys are ofthe following formulae: KCEPDDPWPQCI SEQ ID NO: 31 ACKRNHRWGACV SEQ IDNO: 32 ECGSHAWGRRCK SEQ ID NO: 33


20. The method according to claim 8, wherein said mimotope decoys offormula (I) wherein X_(a) represents the sequence of the followingformula:(X₁₄)_(n14)-X₁₅-X₁₆-H-X₁₇-W-G-X₁₈-(X₁₉)_(n19) in which: n14 and n19independently of one another represent 0 or 1, X₁₄ to X₁₉ represent anyamino acid.
 21. The method according to claim 20, wherein mimotopedecoys are characterized as: X₁₄ represents K, X₁₅ represents R, or G,X₁₆ represents N, or S, X₁₇ represents R, or A, X₁₈ represents A, or R,X₁₉ represents R.
 22. The method according to claim 19, wherein saidmimotope decoys are of the following formulae: ACKRNHRWGACV SEQ ID NO:34 ECGSHAWGRRCK SEQ ID NO: 35


23. The method according to claim 1, wherein said peptide decoyscorrespond to epitopes recognized by antibodies present in saidproteins, the peptide sequence of which is modified by suppression,substitution, or addition of at least one amino acid, said decoys beingdesignated epitope variants.
 24. The method according to claim 23,wherein said epitope variants are of factor VIII.
 25. The methodaccording to claim 24, wherein said epitope variants of factor VIII areof the following formula (II): (SEQ ID NO: 49)(C)_(n1)-X₁-X₂-X₃-L-T-D-S-E-M-D-V-V-R-X₄-X₅-(C)_(n2)

in which: n1 and n2 independently of one another represent 0 or 1, X₁ toX₅ represent any amino acid. with the exception of the peptide offormula DDDLTDSEMDVVRFD (SEQ ID NO: 36).
 26. The method according toclaim 25, wherein said epitope variants are characterized as: X₁represents D or A, X₂ represents D or A, X₃ represents D or A, X₄represents F, Y, or W, X₅ represents D or A.
 27. The method according toclaim 26, wherein said epitope variants are of the following formulae:DDDLTDSEMDVVRFD SEQ ID NO: 37 DDDLTDSEMDVVRYD SEQ ID NO: 38DDDLTDSEMDVVRWD SEQ ID NO: 39 CDDDLTDSEMDVVRFDC SEQ ID NO: 40AAALTDSEMDVVRFA SEQ ID NO: 41


28. A pharmaceutical composition characterized in that it comprisesdecoys peptide or pseudopeptide decoys binding to antibodies capable ofbeing developed against endogenous proteins within the framework ofautoimmune pathologies or against exogenous proteins administered to thepatients, in particular within the framework of pathologies due to adeficiency in these proteins, the amino acid sequence of said peptide orpseudopeptide decoys differing from that of the epitopes recognized bysaid antibodies in combination with pharmaceutically acceptablevehicles.
 29. A peptide sequence of formula (I) having mimotope decoysof factor VIII have the following formula (I):(X₁)_(n1)-C-X_(a)-C-(X₂)_(n2) in which: n1 and n2 independently of oneanother represent 0 or 1, X₁ and X₂ independently of one anotherrepresent a natural or non-natural amino acid, X_(a) represents a chainof 8 to 10 natural or non-natural amino acids
 30. A peptide sequence offormula (II) having epitope variants of factor VIII are of the followingformula (II): (SEQ ID NO: 49)(C)_(n1)-X₁-X₂-X₃-L-T-D-S-E-M-D-V-V-R-X₄-X₅-(C)_(n2)

in which: n1 and n2 independently of one another represent 0 or 1, X₁ toX₅ represent any amino acid with the exception of the peptide of formulaDDDLTDSEMDVVRFD (SEQ ID NO: 36)
 31. A product comprising: at least onepeptide sequence of formula (I), and/or at least one peptide sequence offormula (II), and at least one compound chosen from the extracted, orrecombinant factor VIII, or other derivatives of human or animal origin,as combination products to be used simultaneously, separately or spreadover time, in factor VIII substitution therapy, within the framework ofthe treatment or prophylaxis of hemorragic accidents in patientssuffering from hemophilia A.